In a recent study published in PNAS, researchers investigated the association between the increased levels of anti-platelet factor 4 (PF4) antibodies and the clinical complications of coronavirus disease 2019 (COVID-19).
The anti-PF4 antibodies are analogous to the pathogenic antibodies found in patients with heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombosis with thrombocytopenia (VITT). Both HIT and VITT are fatal syndromes characterized by thrombosis and thrombocytopenia. Progression to severe COVID-19 is also frequently associated with thrombosis and thrombocytopenia.
The main pathological finding in severe COVID-19 is diffuse microvascular thrombosis. It restricts the blood supply to parenchymatous organs resulting in multiple organ failures. Thus, these are detected invariably in the lungs and other organs of deceased COVID-19 patients, suggesting that coagulation abnormalities are common in severe COVID-19 patients.
About the study
In the present study, researchers enrolled 100 patients admitted to the Johns Hopkins Hospital for moderate or severe COVID-19 between April 2020 and April 2021. They had a maximum World Health Organization (WHO) disease severity score between four and ten during hospitalization. All these patients were unvaccinated for SARS-CoV-2 and heterogenous in age, ethnicity, gender, and comorbidities. Since many were critically ill, 47% of these enrollees had to be given organ support in the intensive care unit (ICU) during hospitalization, and 19 died of COVID-19 complications.
The team assessed the ability of patient serum to induce platelet activation using a flow cytometry assay that revealed surface exposure of P-selectin (CD62P). The primary outcome variable in this study was the level of anti-PF4 antibodies in serum measured once during hospitalization. For platelet reduction assessments during hospitalization, the researchers computed the difference between the maximum and minimum counts before and within 30 days after the sampling date, respectively, for each patient.
The team analyzed the association between anti–PF4 antibody levels and multiple demographic, clinical, and laboratory variables depending on the covariate. They used linear regression for continuous and ANOVA for categorical variables and a two-tailed t-test if the covariate represented two sample groups. Finally, the researchers used multiple regression analysis to evaluate the association between anti-PF4 antibodies and maximum disease severity score after adjusting for age, race, body mass index (BMI), and unfractionated heparin (UFH) treatment.
The prevalence and the levels of anti-PF4 antibodies in healthy blood donors were very low. On the contrary, the authors noted anti-PF4 antibodies in the serum of 95% of hospitalized COVID-19 patients, with a mean optical density (OD) value of 0.871 ± 0.405 standard deviations (SD). The OD value was higher than the potential threshold of clinical significance (i.e., 0.75) in 65% of patients, while it was >10 for 28.0% of patients. The OD level of anti-PF4 antibodies was also significantly higher in hospitalized patients with COVID-19 than in convalescent COVID-19 patients. Likewise, 64.3% of patients hospitalized for severe acute respiratory disease (ARD) showed a high rate of anti-PF4 antibody positivity.
The study findings confirmed that low levels of anti-PF4 antibodies could occasionally be present in healthy individuals. Thus, the high frequency of low-level anti-PF4 antibodies detected in patients with COVID-19-unrelated severe ARD reflects non-specific activation of innate immunity with no relevance to disease pathogenesis.
The UFH is a standard prophylaxis in severe COVID-19 cases with thrombotic complications. The UFH-treated patients in the current study cohort included the most severe cases, 23 of 27 had a disease score of 9 or 10. Yet, 47% of patients developed anti-PF4 antibodies without receiving any form of heparin for at least six days before sampling, indicating that exposure to heparin did not induce anti-PF4 antibodies. Also, the dosing method and UFH dosage did not impact the anti-PF4 levels.
The authors also did not observe a correlation between the prevalence of anti-PF4 antibodies and the patient’s age, BMI, or pre-existing comorbidities. However, their prevalence was higher in male than female patients and African Americans or Hispanic ethnicities than in other ethnic groups.
In HIT and VITT cases, induction of anti-PF4 antibodies typically takes seven to 10 days after heparin administration or adenovirus-vectored vaccines, respectively. Intriguingly, the authors noted that the time taken in the induction of anti-PF4 antibodies coincided with the appearance of severe COVID-19-related complications following the initial acute phase. Though thrombotic events, such as pulmonary embolism, were relatively infrequent (occurred only in 12% of patients in the study cohort), their appearance was not associated with the anti-PF4 antibody levels. In addition, sera from hospitalized patients with COVID-19 showed increased platelet-activating activity. Yet, it was markedly lower than HIT and VITT patients and did not correlate with the anti-PF4 antibody levels. Moreover, the precise mechanism governing the clinical significance of low-level platelet activation in the serum of COVID-19 patients remains unclear.
Previous studies focused on immunoglobulin G (IgG) testing, the prevalent isotype detected in the HIT and VITT syndromes. The researchers in the current study documented a multi-isotype anti-PF4 antibody response in severe COVID-19 patients, who show a high prevalence of IgM isotype. Thus, studies should address these discrepancies. The mechanism governing the generation of anti-PF4 antibodies in COVID-19 patients is currently unknown. Preliminary studies have suggested that PF4 directly interacts with the SARS-CoV-2 spike (S) protein leading to the formation of multimolecular aggregates encompassing PF4 and S proteins, similar to those elicited by heparin.
Antigenic modifications of PF4 induced upon binding to the SARS-CoV-2 S might, in turn, expose cryptic immunogenic epitopes in the chemokines recognized by the immune system. These possibilities are under investigation in small animal models. Whether the S proteins of different SARS-CoV-2 variants interact with PF4 with the same affinity remains unknown. Thus, it will also be interesting to evaluate if infection with different SARS-CoV-2 variants with reduced pathogenicity would trigger similar rates of anti–PF4 antibody induction.
To summarize, the levels of anti-PF4 antibodies detected in the majority of COVID-19 patients were over the threshold for clinical significance, suggesting a specific mechanism of induction of such antibodies with potential pathological consequences. The in vitro platelet activation assays used in the current study could not accurately recapitulate the in vivo complexities observed in COVID-19 patients.
Yet, the role of anti-PF4 antibodies in the pathogenesis of COVID-19-related vascular complications is plausible; thus, future studies should investigate the same. Further mechanistic studies validating the role of anti-PF4 antibodies in the COVID-19 pathogenesis could guide the management of severely ill COVID-19 patients.